Polysaccharides are extremely abundant and diverse, including structures such as cellulose, chitin, starch, and glycogen, to name just a few examples. Glycosylation is important not only to the formation of carbohydrates, but also glycoproteins, and glycolipids, cellular processes including protein folding, trafficking, recognition, and stability, cell growth, migration, communication, and immune response. It also plays a role in the virulence of many microorganisms.
The formation of carbohydrate molecules in biological systems depends upon glycosyltransferase enzymes, which perform the key function of transferring a monosaccharide or an oligosaccharide from a donor molecule to an acceptor. Given the abundance and structural diversity of carbohydrates, it is not surprising that there are numerous glycosyltransferase enzymes, each of which are specific to particular donor molecules and acceptor molecules. For example, it is estimated that the human genome encodes more than 200 glycosyltransferase genes.
Glycosyltransferase enzymes add sugar groups not only to polysaccharides but also to proteins and lipids to form, glycoproteins, and glycolipids. Most glycosyltransferases use activated nucleotide sugars as donor molecules. These glycosyltransferases are referred to as Leloir enzymes. A Leloir glycosyltransferase transfers the sugar group from a sugar nucleotide to a substrate molecule (e.g., sugar, protein or lipid), generating a nucleotide leaving group. The other glycosyltransferases, referred to as non-Leloir enzymes, use other donors such as lipid phosphosugars, resulting in leaving groups such as lipid phosphates.
Because of their key roles in development, cellular functions, and pathogenicity, it is desirable to monitor glycosyltransferase activity. For example, glycosyltransferases may be ideal targets for drug therapies, to promote, inhibit, or block their activities. A method of monitoring glycosyltransferase activity would therefore be very useful in the development of such drugs, among other uses.
Several methods have attempted to measure glycosyltransferase activity, but each of these methods suffers from certain difficulties which limit their usefulness. For example, one traditional method of assaying glycosyltransferases uses radioisotopes. However, besides involving the use of radioactivity, this method requires the use of a specific labeled compound and separation of the donor from the acceptor, which is a time consuming process. Thin-layer chromatography (TLC), high pressure liquid chromatography (HPLC) and LC-MS can also be used. However, these methods are limited by the lack of speed, expensive reagents and equipment and the high level of expertise which is required to perform them. Antibody labeling and fluorogenic compounds can also be used, with the antibodies and fluorogenic compounds specifically targeted to the nucleotide products. While these methods do not require separation of the donor and acceptor, the specific antibodies and fluorogenic compounds can be difficult to produce, resulting in high cost, and can entail long assay times and high background readings, making the results less sensitive than desired.